Clinical use of allogenic skin has been limited by short graft survival times attributable to the immunologically mediated rejection. While long-term graft survival has been achieved in the case of solid organ transplants for many years using an arsenal of immunosuppressive agents, there has been no corresponding improvement in the acceptance of skin allografts.
A concerted effort to develop semi-synthetic skin equivalents has been undertaken over the years. Some of these technologies have been commercialized including an artificial dermis such as Integra (Integra LifeSciences Corporation, Plainsboro, NJ) followed by the application of sheets of autologous cultured cells once circulation has been established. Alloderm (LifeCell, Woodlands, TX) consisting of human dermis rendered antigen free over which a thin layer of skin graft can be applied. Other products include Adaptec (J&J, New Brunswick, NJ), Conformat 2 (Smith & Nephew, Hull, UK), Epicell (Genzyme Biosurgery, Cambridge, MA), and Laser Skin (Fidia Advanced Biopolymers, Abano Terme, Italy). The disadvantages of these therapies remain the vulnerability of the epidermal layer and the lack of appendages such as hair follicles or sweat glands that breach both the epidermal and dermal layers. Use of synthetic skin equivalents has been associated with high rates of infection and graft loss, highlighting the need for a dermal component.
While an autograft remains the preferable treatment for skin lost from burn or trauma, long-term acceptance of an allograft represents the next best alternative. The ability to overcome the immunologic barriers that have historically resulted in the failure to achieve long-term acceptance of skin allografts could have a corresponding major overall impact in the field of clinical transplantation. A new approach that could potentially improve both skin and solid organ allograft survival would be an immunomodifying therapy that is tissue-specific. Most efforts to prolong allograft survival have focused on the effector arm of the immune system. With the exception of pretransplant graft treatments such as the depletion of passenger leukocytes, efforts have focused on interrupting the effector cell cascade. Our approach to tissue-specific allograft immunomodification entails coating the wound surfaces of the allograft skin with a nano-barrier membrane (NB-LVF4). Similarly, we have used the NB-LVF4 for the immunomodification of the vascular surfaces within a solid organ allograft.1 The goal is to interrupt the normal interface by providing a physical barrier between recipient and donor tissues.
NB-LVF4 is composed of an array of laminin, vitrogen, fibronectin, and type IV collagen that provide an apical surface that remains nonthrombogenic and nonimmunogenic. The network of molecules constituting the NB-LVF4 functions as a barrier to immune cell migration, while simultaneously allowing the free diffusion of nutrients and oxygen. In a previous study, we reported that NB-LVF4 used as an artificial interface between a skin allograft and the wound surface prolonged graft survival in the absence of systemic immunosuppression.2 In this study, we evaluated whether the nano-barrier membrane could function as a targeted drug delivery system. In this feasibility study, we investigated whether incorporation of fibroblast growth factor-1 (FGF-1) into the membrane before application could further improve skin allograft acceptance. The selection of FGF-1 was based on its known function as a wound hormone, with the potential of reducing both the time the skin allograft would be dependent on plasmatic circulation and accompanying inflammation.
Materials and Methods
Nano-Barrier Membrane (NB-LVF4)
The NB-LVF4 used for immunocloaking is a proprietary technology of Breonics, Inc., (Albany, NY). NB-LVF4 is comprised of laminin, vitrogen, fibronectin, and type IV collagen made from proteins synthesized by cultured corneal endothelial cells. The components are polymerized into a three-dimensional transparent membrane approximately 200 nm in thickness. Laminin polymers serve as the template for the NB-LVF4 assembly. The cross-linked type IV collagen provides the structural integrity and the disulfide bonds stabilize the components in the membrane.
Nonimmunogenicity of NB-VLF4
Mixed Lymphocyte-Vascular Endothelial Cell Reaction.
The mixed lymphocyte-vascular endothelial cell reaction (MLER) was performed using allogenic peripheral blood mononuclear cells (PBMNC) as the responding cell population with vascular endothelial cells (VEC) as the stimulator. The VEC were isolated from blood vessels using collagenase digestion, seeded into tissue culture plates, and maintained in culture until confluent. The PBMNC were isolated from whole heparinized blood on Ficoll-Hypaque gradients. The PBMNC were collected, washed twice, and resuspended at a concentration of 1 × 106/ml. Once confluent, 2.5 × 105 of responding allogenic PBMNC were added to each of the triplicate well. The cultures were maintained at 37°C for an additional 4 days, pulsed with 0.6 μCi 3H-thymidine, and harvested 18 hours later. DNA synthesis was evaluated by scintillation counting of the harvested cells. In the wells where NB-LVF4 was tested, the cold solubilized membrane was neutralized and then applied to cover either the bottom of the wells or to cover the confluent layer of VEC.
The test combinations included are listed below:
- Negative control: PBMNC alone
- Positive control: PBMNC + allogenic VEC
- Test: PBMNC + NB-LVF4 treated allogenic VEC
- Test: PBMNC + NB-LVF4 alone
The testing was performed in triplicate on two separate days and the results represented as the mean of the counts per minute (CPM). The Stimulation Index (SI) was calculated as follows: Mean CPM Test/Mean CPM Negative Control. The effectiveness of NB-LVF4 treatment was determined by the % of inhibition it provided in comparison to the positive controls.
Application and Transplantation Procedure
The process involved in solubilizing and then polymerizing the NB-LVF4 membrane at physiologic temperature has been previously described.2 A thin continuous membrane that covers the surface of the wound and the basolateral surface of the skin allograft resulted. In the test group where FGF-1 (Breonics, Inc., Albany, NY) was added to the membrane, 5 μg was added to the cold solubilized mixture just before treating the allograft and wound bed. As NB-LVF4 is permeable to low-molecular-weight compounds, free transport of nutrients and oxygen is unaffected and the tissue remains viable. Likewise, the membrane supported cellular functions similar to the role of extracellular matrices in substrata tissues.
Genetically inbred BALB/c and C57BL/6 mice were obtained from Charles River Company (Wilmington, MA). The BALB/c-C57BL/6 model is considered to represent an excellent model for skin allograft transplantation because of the major H-2 complex incompatibilities. Congenic BALB/c mice possess the d phenotype while congenic C57BL/6 possess the b phenotype.3 Mice were housed in compliance according to our Institutional Animal Care and Use Committee following the “Principles of Laboratory Animal Care” with free access to food and water. Full-thickness 8-mm skin grafts were cross-transplanted between the two genetically unrelated groups of mice (BALB/c and C57BL/6 mice) and sutured in place. Control skin allografts were transplanted without treatment after 15 minutes incubation at 37°C in a sterile milieu. In the test skin allografts, the 8-mm skin grafts with 100 μl of the neutralized and normothermic NB-LVF4 with and without FGF-1 were carefully layered on to the subdermal surface of the skin graft using a sterile syringe. Treated skin grafts were then incubated at 37°C for 15 minutes. During the incubation period, an additional 100 μl of the neutralized and solubilized membrane was also layered onto the wound bed on each mouse. After polymerization, the treated skin grafts were allotransplanted onto the surface of the already treated wound bed by suturing the graft in place.
The animals were followed by daily visual inspection as previously described.2 The occurrence of rejection was defined as graft necrosis >90%, resulting in the loss of viable skin.
Group 1 (n = 15): Cross-transplants of untreated skin grafts between BALB/c and C57BL/6 mice.
Group 2 (n = 15): Cross-transplants of NB-LVF4 treated skin grafts between BALB/c and C57BL/6 mice.
Group 3 (n = 15): Cross-transplants of NB-LVF4 + FGF-1 (5 μg) treated skin grafts between BALB/c and C57BL/6 mice (Figure 1).
Immunologic Screening by Flow Cytometric Cross-match
The development of donor-reactive antibodies was determined by flow cytometric cross-matching to demonstrate the immunologic basis for graft loss. The flow cytometric cross-match, a sensitive test for determining immunologic status, was performed by incubating the recipient sera with the donor lymphocytes stained with a fluorochrome-conjugated secondary antibody [fluorescein isothiocyanate-conjugated goat anti-mouse IgG (H and L); Jackson Immuno Research, West Grove, PA]. Specifically, the target lymphocytes were isolated from either the spleens of naive BALB/c and C57BL/6 mice or the cross-transplanted donors after euthanasia. The lymphocytes were used at a working concentration of 2 × 105 cells that were incubated with 50 μl of recipient serum for 30 minutes, washed, and resuspended in 10 μl of fluorochrome-conjugated secondary antibody. After a second 30-minute incubation, the lymphocytes were again washed followed by fixation in paraformaldehyde. The fixed cells were then analyzed using a FACScan (Becton Dickinson, Franklin Lakes, NJ) and Cellquest software (Cellquest Inc., Largo, FL). The baseline channel fluorescence was determined using negative controls consisting of both autologous sera and sera from naive mice.
The median channel fluorescence (MCF) shift used for determining positivity was calculated as the SD based on the average of the median channel fluorescence of the controls. The cutoff for positivity was set at 3SD. The presence of donor-reactive antibody was assigned when the MCF shift was greater than the established cutoff for positivity.
Effect of FGF on B Cell Activation
A modified MLER was performed using FGF-1 treated and untreated allogenic PBMNC as the responding cell population with VEC as the stimulator as described above. The cultures were maintained at 37°C for 24 hours and the CD20+ B cells were then counted. CD20 is a marker for B cells as it is constitutively expressed on all B cells. All testing were performed in triplicate.
Control Group (n = 14): Untreated PBMNC.
Test (n = 14): Treated PBMNC incubated vol/vol with 5 μg of FGF-1 for 60 minutes at 37°C before application to the VEC.
Immunohistochemical Analysis of CD25+ B Cells
The CD20+ B cells from each of the triplicate wells of both control and test groups were evaluated for CD25 expression as a marker for B cell activation. An indirect immunofluorescence assay was performed using a murine monoclonal anti-CD25+ antibody (VMRD, CO) incubated with the CD20+ B cells for 30 minutes at 37°C. After incubation, the cells were washed twice and then incubated with a rabbit anti-mouse IgG (Sigma, MO) for an additional 30 minutes. The cells were again washed twice, in resuspended and evaluated for the number of with cells with the phenotype of CD20+, CD25+ using a Victor2 fluorescence reader (PerkinsElmer Life Sciences).
All data ares represented as the mean with accompanying SD for each experimental group. A t test analysis was used to determine statistical significance of the experimental findings.
Mixed Lymphocyte-Vascular Endothelial Cell Reaction
The traditional mixed lymphocyte culture has been long used as sensitive test for evaluating cellular compatibility within the MHC class II complex in transplantation (Table 1). The results were interpreted as “mini-graft” where a SI of <5.0 was considered negative, indicating that there was no significant proliferative response to the allogenic cells. Similarly, a SI >5.0 was considered positive where an aggressive immunogenic response could be anticipated. In this study, VEC were used as the stimulator cell in place of lymphocytes. The PBMNC proliferated when stimulated by allogenic VEC, resulting in a SI of 62.0. However, when NB-LVF4 was applied to the surface of the confluent VEC, the PBMNC proliferation was essentially eliminated with a 98.1% inhibition of the proliferative response to the VEC. Support for the nano-barrier being nonimmunogenic is the observation that PBMNC did not respond to NB-LVF4 alone. Therefore, NB-LVF4 is not immunogenic, does not elicit a proliferative response of itself, and results in an immunocloaking effect.
Primary Skin Graft Survival
Rejection occurred in the group 1 mice receiving the untreated skin allografts with a mean of 7 (±1.5) days. Rejection of the untreated skin grafts resulted in open wounds that eventually formed scabs. In contrast, pretreatment of the skin allografts with NB-LVF4 was found to substantially prolong allograft survival (p < 0.05) with rejection occurring with a mean of 27 (±3.8) days. The observed prolongation of graft survival in group 2 recipients reached statistical significance. In all cases, the treated groups demonstrated good hair regrowth, which in some cases exceeded surrounding native hair regrowth. The eventual rejection of the NB-LVF4 treated skin allografts did not result in an open wound or in the formation of a scab, but the skin graft was eventually sloughed, leaving an intact pseudodermis. Allograft survival was also prolonged in group 3 mice treated with FGF-1, with rejection occurring with a mean of 28 (±2.4) days (p < 0.05).
The results of the flow cytometric analysis indicated that the group 1 recipients of untreated skin allografts demonstrated a 10-fold shift in the number of CD4+ cells (Table 2). In contrast, in both group 2 treated and group 3 treated with the addition of FGF-1, the shift in the number of CD4+ cells was significantly reduced with a fourfold increase.
By 14 days post-transplantation, all the control recipients in group 1 demonstrated donor-reactive antibody, indicating an immunologic basis for the skin graft loss. Donor-specific antibody was also found to have developed in the group 2 recipients of NB-LVF4 treated skin by 45 days post-transplantation. However, the recipients of treated skin containing FGF-1 did not develop donor-specific antibody.
FGF Effect on B Cell Activation
Exposing naïve B cells to FGF-1 before stimulation with allogenic VEC resulted in significant inhibition of B cell activation (Figure 2). The number of B cells staining positive for the CD25 phenotype was reduced to just 8% of the expression observed in the untreated controls.
Our approach to graft immunomodification entails coating the wound surfaces of the allograft skin with a nano-barrier membrane (NB-LVF4).2 The goal is to interrupt the normal interface by providing a physical barrier between recipient and donor tissues. The NB-LVF4 membrane is composed of laminin, vitrogen, fibronectin, and type IV collagen that provides an apical surface that remains nonthrombogenic and nonimmunogenic. The results from the MLER studies demonstrate that the NB-LVF4 membrane is nonimmunogenic and treatment of allogenic VEC with the membrane prevents the proliferative response that normally occurs in its absence.
Attempts to immunomodify allogenic tissues and organs with the goal of eliminating systemic immunosuppression is not new and have been ongoing for more than 20 years.4–7 A variety of encapsulating materials made from biocompatible polymers have been used for immunomodifying a number of proteins and isolated cells. Of these polymers, the most widely studied is poly(ethylene-glycol) (PEG), which has been covalently bonded to a variety of proteins, carbohydrates, lipids, and cells since the 1970s. PEG has been used in several applications, including reducing red blood cell antigenicity in both blood group mismatched and xenotransfusion, coupled with hemoglobin to develop blood substitutes, and treatment of isolated pancreatic islets.8–11 While red blood cells (RBC) PEGylation is useful for some blood group antigen coverage, the ABH antigens A and B have been difficult to mask.12–15 Interestingly, mPEG has been shown to be stably attached to RBC membranes for 30 days, even in the case of hemolysis.16
The addition of a variety of factors to the NB-LVF4 membrane before polymerization could present the opportunity to further improve the outcomes. As a first step toward this goal, this study evaluated whether the addition of FGF-1 would further improve skin allograft survival. As the goal of this study was to establish feasibility, biopsies were not taken because of concerns that it would disrupt the membrane integrity and initiate rejection.
FGF-1 can be thought of as a wound hormone that has angiogenic properties, stimulates DNA synthesis, regulates cell growth, and inhibits apoptosis. In essence, FGF signaling regulates the activity of virtually all higher vertebrate cell types.17 FGF signal transduction is medicated by a family of membrane tyrosine kinases through both high- and low-affinity FGF receptors, allowing for a number of signaling cascades.18
The results of this initial feasibility study demonstrate that treatment with NB-LVF4 supplemented with FGF-1 delays the onset of acute skin allograft rejection when compared with untreated controls. A surprising result was the observation that the addition of FGF-1 appears to prevent the development of donor-specific antibody. This observation is consistent with a report where FGF-deficient mice were shown to produce increased levels of antibody.19 Conversely, antibody production was found to be suppressed in FGF overexpressing mice.
We evaluated whether FGF-1 mediated the observed inhibition of antibody production by interfering with B cell activation using CD25 as a marker. CD25 is the alpha chain of the IL-2 receptor that is a transmembrane protein expressed on activated B cells. CD25 expression on B cells is selectively upregulated by Toll-like receptor 2 (TLR2), TLR4, and TLR9 ligands. Blockade of the nuclear factor (NF)-κβ prevents CD25 upregulation in B cells. Such blockade of CD25 has been shown to interfere with the proliferative response in the MLR.20 CD25+ B cells secrete significantly increased levels of IL-10 when compared with CD25− B cells and serve as memory cells that enhance the immune response.21 Most importantly, CD20+ and CD25+ B cells have been shown to produce increased levels of immunoglobulins.22 These early findings suggest that FGF prevented B cell activation significantly to result in the prevention of donor-specific antibody development.
These results provide further evidence for a role of NB-LVF4 in providing temporary protection from skin allograft rejection. Our results also demonstrate that NB-LVF4 can be used as a targeted drug delivery system to immunomodify skin allografts. The inclusion of FGF-1 within the NB-LVF4 appears to have prevented B cell activation and interfered with their ability to produce donor-specific antibody. Such protection would appear logical during the phase when graft survival is dependent on plasmatic circulation and during the early period of communication between recipient and donor microvessels. What is not yet understood is how NB-LVF4 provides protection, leading to prolongation of graft survival when mature complex capillaries, arterioles, and venules should have developed (>7 days postengraftment). The goal of future studies will be to study the fate of NB-LVF4 over time and to determine its mechanism of protection.
Supported by the National Institutes of Health (NIH), R3AI42487A.
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